Project Summary West Nile virus (WNV) is an emerging, neurotropic virus of the Flavivirus genus that is transmitted to humans via infected mosquitoes. Flaviviruses include globally important pathogens, such as dengue (DENV) and Zika (ZIKV) virus, which infect hundreds of millions yearly. Currently, there are no specific antiviral treatments for any flavivirus. Given the spread of flaviviruses across the globe and the dearth of options to prevent or treat them, it is imperative that we develop a better understanding of host processes that impact infection. We used affinity purification and mass spectrometry to identify the physical interactions that occur between WNV and host proteins. In collaboration with Nevan Krogan, we utilized data from parallel studies of DENV and ZIKV to focus on host proteins targeted by multiple flaviviruses. We discovered 259 high-confidence WNV-interacting host proteins; of those, 49 host proteins interacted with the analogous viral protein in either DENV or ZIKV. This analysis revealed that the most significant overlap was for capsid- and NS5- interacting proteins. To define the shared interactors that are important for infection, we employed an RNAi screen in the context of WNV, DENV and ZIKV infection. We found 23 factors that impacted WNV infection, 12 that impacted WNV and one additional flavivirus, and 8 factors influencing infection of all three flaviviruses. Among these, we identified two host proteins with roles in antiviral signaling and cell-intrinsic immunity. USP15 is a host deubiquitylase that promotes WNV infection and interacts with flavivirus NS5 proteins. We show USP15 is required for WNV infection and acts as a negative regulator of Type I interferon in this context. We will establish the role of the WNV NS5-USP15 interaction in this phenotype by identifying the residues in USP15 that are critical for interaction with NS5 and generating NS5-binding deficient USP15 mutants to test in our studies. Activation of the Type I interferon response requires ubiquitylation of RIG-I, and we propose that USP15 inhibits Type I interferon signaling via deubiquitylation of RIG-I. To test this, we will monitor activation and ubiquitylation of RIG-I upon knockdown of USP15 and determine the role of the USP15- NS5 interaction in this response. We have also identified the flavivirus capsid-interacting host protein WIBG, which restricts WNV infection. WIBG is involved in RNA regulatory processes, including nonsense-mediated RNA decay (NMD) and interaction with the exon-junction complex (EJC) proteins, Y14 and MAGOH. We show that NMD is inhibited and that the interaction of WIBG with Y14/MAGOH is disrupted in flaviviral infection. Moreover, we demonstrate that NMD and the EJC are antiviral, as depletion of a canonical NMD factor, UPF1, and an EJC protein, MAGOH, results in increased flavivirus infection. We propose that NMD is an antiviral host process that is antagonized by flaviviruses. We will determine the mechanism by which NMD inhibits WNV infection and determine how the WIBG-capsid interaction influences this process. The goal of this proposal is to uncover the mechanisms by which flaviviruses subvert host innate antiviral mechanisms.